Microbiological process for preparation of internal monoalkenes

ABSTRACT

A resting-cell suspension of glucose-grown Nocardia salmonicolor cells will oxidize C14-C20 alkanes to internal monoalkenes having a cis configuration. Dehydrogenating an individual alkane produces a mixture of monoalkenes with one alkene predominating. Dehydrogenation is limited to the internal carbon atoms of long chain alkanes, predominantly the carbon atoms near the middle of the molecules. The oxidation of hexadecane by Norcardia salmonicolor produces a mixture of olefins comprising cis-7hexadecene, 80 percent; cis-8-hexadecene, 18 percent; and cis-6hexadecene, 2 percent.

United States Patent lnventors Appl. No.

Lester E. Casida, Jr.

State College, Pa.;

Bernard J. Abbott, Edison, NJ.

Nov. 9, 1970 Dec. 21, 1971 Texaco Inc.

New York, N.Y.

Continuation-impart of application Ser. No. 736,563, June 13, 1968, now abandoned. This application Nov. 9, 1970, Ser. No. 88,192

MICROBIOLOGICAL PROCESS FOR PREPARATION OF INTERNAL MONOALKENES 13 Claims, No Drawings US. Cl 195/28 Int. Cl Cl2d 13/00 Field of Search 195/28, 51

[56] References Cited UNITED STATES PATENTS 3,383,289 5/1968 Raymond et al 195/28 Primary Examiner A. Louis Monacell Assistant Examiner-Seymour Rand Attorneys-Thomas H. Whaley and Carl G. Ries ABSTRACT: A resting-cell suspension of glucose-grown Nacardia salmonicolor cells will oxidize C,,-C,,, alkanes to internal monoalkenes having a cis configuration. Dehydrogenating an individual alkane produces a mixture of monoalkenes with one alkene predominating. Dehydrogenation is limited to the internal carbon atoms of long chain alkanes, predominantly the carbon atoms near the middle of the molecules. The oxidation of hexadecane by Norcardia sulmom'color produces a mixture of olefins comprising cis-7-hexadecene, 80 percent; cis-8-hexadecene, 18 percent; and cis-o-hexadecene, 2 percent.

MICROBIOLOGICAL PROCESS FOR PREPARATION OF INTERNAL MONOALKENES CROSS-REFERENCE TO RELATED APPLICATION This is a continuation-in-part of application Ser. No. 736,563, now abandoned.

BACKGROUND OF THE INVENTION The process of our invention relates to the production of monoalkenes, particularly those wherein the double bond does not occur on the terminal carbon atom. For convenience, hereinafter, these monoalkenes will be referred to as internal monoalkenes. The process of our. invention is more particularly related to a process employing a micro-organism to selectively dehydrogenate the internal carbon atoms of alkanes to produce monoalkenes.

It is known that the initial intermediates from the microbial oxidation of alkanes are alcohols, ketones, hydroperoxides or terminally unsaturated alkenes. Anaerobic dehydrogenation of heptanes has produced l-heptene. Cells of the genera Nocardia, Pseudomonas, Rhodotorula and Microococcus will produce l-hexadecene as an intermediate in an aerobic oxidation of hexadecane.

To date, internal monoalkeneshave not be observed among the products of microbial hydrocarbon oxidation.

SUMMARY OF THE INVENTION We have found that a particular micro-organism will oxidize alkanes to produce internal monoalkenes. More particularly, we have found that a resting-cell suspension of glucose-grown Nocardia salmonicolor cells will oxidize a C -C, alkane or mixtures thereof producing substantial quantities of monoalkenes have a cis configuration and wherein the double bond is not on a terminal carbon atom but on the central carbon atoms. A deposit of this species with the American Type Culture Collection has been assigned the number ATCC 21243. The process is carried out as a two-stage fermentation with the cells grown first on a carbohydrate-containing medium (the growth stage), then separated from this medium and added as a nongrowing resting-cell preparation to a mixture of water or buffer and hydrocarbon (the conversion stage). The growth stage usually consists of two separate parts, an inoculum growth phase, and a cell production growth phase. The second portion of the fermentation process is the alkane-to-alkene conversion stage.

The inoculum for cell production may be grown on a glucose-DMS medium seeded with a loop transfer of stock culture of Nocardia salmom'color being maintained on nutrientagar containing 0.1 percent glucose. In the cell production growth phase the presence of iron salt in the aqueous growth medium is critical as regards the reproducibility and amount of conversion occurring in the subsequent hydrocarbon conversion stage. The temperature and pH conditions during this growth phase are also critical in that they influence the enzymatic activity in the conversion stage, although the temperature is less critical when the concentration and valence of the iron present in the growth medium are carefully controlled. The conversion stage requires oxygen and is temperature dependent.

The monoalkenes produced by our process have a variety of uses. They can serve as intermediates for the production of (a) monocarboxylic acids via oxidative cleavage, (b) alcohols via the 0x0 reaction, (c) alkylbenzenes via alkylation, (d) epoxides and (e) unsaturated sulfonic acids via reaction with 80;. These products in turn can be used to make plasticizers, detergents and other surface-active agents.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In accordance with the present invention, a resting-cell suspension of glucose-grown Nocardia salmom'color cells oxidize a C C, alkane or mixtures thereof to a mixture of cis internal monoalkenes.

The particular micro-organism utilized in our process to dehydrogenate alkanes is a species known as Nocardia salmonicolor. We have found a particular strain of this species to be especially useful and have designated it as strain PSU-N-l 8. This micro-organism was deposited with the American Type Culture Collection, Rockville, Maryland, on May 6, I968 where it has been given the designation, ATCC 21243, and where it is presently available for distribution to the public without reservation, having been released for distribution without reservation as of May 1, 1970. This strain of N. salmonicolor grows well on either glucose or hexadecane as a carbon substrate. However, only minute quantities of unsaturated hydrocarbons accumulate during growth on hexadecane. Similar results are obtained during resting-cell incubation of hexadecane-grown cells with hexadecane. However, dehydrogenation of alkanes by N. salmonicolor may be carried out as a two-stage fermentation with the cells grown first on a carbohydrate-containing medium, then separated from this medium and added as a nongrowing resting-cell preparation to a mixture of water or buffer and alkane substrate. Thus this fermentation is ideally composed of three separate steps: an inoculum growth step, a cell production growth step and an alkane-to-alkene conversion step. For simplicity, these steps will be hereinafter referred to as the inoculum, growth and conversion steps, respectively.

Stock cultures of Nocardia salmonicolor may be maintained on nutrient-agar slants containing 0.l percent glucose. Sufficient inoculum for cell production may be grown in 4 days in 50 ml. of glucose-DMS broth. A small quantity of this inoculum, for example, 5 rnl., when added to approximately 250 ml. of similar glucose-DMS broth will produce, after incubation of about 5 days of 29 C. on a rotary shaker, sufiicient cell growth to prepare the required resting-cell suspension. The cells from the growth step may be harvested by 'any wellknown technique, for example, centrifuging the growth mixture, recovering the cells and resuspending the cells in a phosphate buffer solution having a pH of 7. This resting cell suspension may then be brought into contact with the alkane feedstock. The desired dehydrogenation will occur after several days under aerobic conditions at a constant tempera ture of about 29 C., for example.

In the inoculum and growth steps an aqueous nutrient medium may be employed to promote the growth of the micro-organism. We have found that a DMS aqueous medium, modified from that of Raymond and Davis, may satisfactorily be employed. The composition of this medium is set forth in table I below.

In addition to the inorganic salts listed in the above table, we have found that additionsof iron salt to the nutrient medium are essential for the formation of the alkene-producing N. salmonicolor cells. Iron sulfates are to be preferred, although when the nutrient medium contains substantial quantities of sulfates other soluble iron salts may be used. N. salmonicolor grows poorly in the absence of iron salts and the resultant cells will not dehydrogenate alkanes to alkenes. Although additions of either ferrous or ferric salts will promote the formation of reasonable amounts of alkene-producing enzyme in the cells, the presence of iron in both valance states will greatly increase the content and/or the activity of the enzymes. Iron salts in the nutrient medium should be present in the following concentrations: ferrous salts, expressed as the sulfate, between about 0.0075 and 0.0l wt. percent, preferably about 0.01 wt. percent and ferric salts, also expressed as the sulfate, between about 0.01 and 0.2 wt. percent, preferably about 0.l wt. percent.

Carbon nutrition may be supplied in the aqueous medium by additions of glucose sufficient to provide a concentration level of approximately 5 wt. percent.

Proper temperature and pH and a supply of air are also essential to the satisfactory growth in both the inoculum and growth steps. Thus the temperature of the aqueous medium should be maintained between 26 and 33 C., and the pH between about 6.8 and 7.2 for a growth period of between about 2 and 5 days. A supply of air is necessary and may be furnished, when growing the micro-organism in 2-liter flasks, by loosely stoppering the flask with cotton and mounting it on a rotary shaker.

The cell growth is initiated in the inoculum step by seeding a small quantity of the nutrient medium with a loop transfer from an agar slant of Nocardia salmonicolor. After several days growth a small quantity of the inoculum may be added to a large flask containing a nutrient medium for cell production. Following incubation for approximately 5 days on a rotary shaker at the preferred temperature, the production of cells is sufficient to permit harvesting and preparation of the testing cell suspension. The cells may be recovered by such means as centrifugation or filtration. Following recovery of the cells they may be resuspended in a neutral buffer solution or in distilled water.

The resting-cell hydrocarbon oxidation is conducted with a volume ratio of resting-cell suspension to alkane of between about 1 to l and about 5 to 1, preferably about 4 to 1. During the oxidation, which is usually accomplished in about 3 to 7 days, the temperature should be maintained between about 23 and 33 C. and the pH between about 6.6 and 7.8. Since the conversion step has an oxygen requirement, agitation is required to bring sufficient quantities of oxygen in contact with the mixture. Although concentrations of iron ions are critical for formation of alkane-dehydrogenating enzyme during the growth of the micro-organism, its addition during the oxidation step would decrease the formation of olefin and is ideally excluded from this step.

The conversion step also can be carried out as inverted phase fermentation, i.e., with an excess of hydrocarbon, and in fact, only small amounts of the aqueous resting-cell suspension are required. Apparently, inorganic salts and other growth nutrients are not required by the resting cells and hence the aqueous nutritive phase and pH control normally employed for hydrocarbon fermentations are not required in the conversion state of the process ofour invention.

Among the alkanes which may be dehydrogenated by the process of our invention are the C to C alkanes, particularly the C to C alkanes.

In the process of our invention, alkene selectivity can be substantial. For example, when dehydrogenating hexadecane with a resting-cell suspension of N. salmonicalor grown on a glucose-DMS medium containing both ferrous and ferric salts, 40-50 percent of the hexadecane can be converted to internal monohexadecenes within a 3-7-day period when using a 4:1 ratio of resting-cell suspension to alkane.

The micro-organism employed in our invention promotes the production of monoolefins having a cis configuration. In addition the double bond is always found near the middle of the carbon chain. A mixture of monoolefins is produced when a pure alkane is employed in our invention, however, the mixture is composed predominantly ofa single olefin. Thus, when hexadecane is oxidized by our process, the mixture consists of 7-hexadecene, 80 percent; 8-hexadecene, 18 percent; and 6- hexadecene, 2 percent. When octadecane is the charge stock, the monoolefins produced comprise 9-octadecene, 91 percent; 8-octadecene, 2-3percent; 7-octadecene, l-2percent and S-octadecene and 6-octadecene in trace amounts. It is interesting to note that in the alkene produced in the greatest yield, the unsaturation occurs on the ninth carbon from one end of the chain, i.e., in the case of 7-hexadecene it is nine carbons from one end and seven from the other while in 9-octadecene it is nine carbons from either end. Apparently the dehydrogenating enzyme has the ability to count off nine carbon atoms from one end of an alkane molecule and dehydrogenate it at that point.

The following examples illustrate the practice of our invention.

EXAMPLE I lnoculum of Nacardia salmonicalor was grown for 4 days in 50 ml. of glucose-DMS medium seeded with a loop transfer of Nocardia salmanicolor (ATCC 21243) from a glucose-nutrient agar slant. 250 ml. of aqueous nutrient solution of table l together with sufficient ferric sulfate to provide a concentration of 0.1 wt. percent were placed in a 2-liter flask and sterilized by autoclaving at 121 C. for 20 minutes. After cooling, glucose was added to provide a glucose concentration in the medium of 5 wt. percent. The inoculum was grown for 4 days at 29 C. under aerobic conditions by incubating the flask containing the micro-organism and nutrient solution on a rotary shaker.

Five milliliters of the inoculum were then added to a 2-liter flask containing 250 ml. of the glucose-aqueous medium containing 0.1 wt. percent ferric sulfate. To promote cell production this flask was incubated for 5 days on a rotary shaker at 29 C. Then the N. ralmonicolor cells were harvested by centrifuging the contents of the flask for 15 minutes. The recovered cells were resuspended in 20 ml. of a 0.1 M phosphate buffer having a pH of 7 to prepare the resting-cell suspension.

This cell suspension was then transferred to a cotton-stoppered 250ml. flask containing 5 ml. of hexadecane and incubated at 29 C. on a rotary shaker for 7 days. At the end of this period, the hydrocarbon layer was sampled and analyzed quantitatively by gas chromatography, supplemented by fatty acid determination, ozonolysis, nuclear magnetic resonance and mass spectroscopy. The results are presented in table ll below.

TABLE ll Dehydrogenation of hexadecane dehydrogenates the alkane at a nonterminal position, predominantly at the central portion of the molecular structure.

EXAMPLE II In a manner similar to that of example I, octadecane was dehydrogenated by Nocardia salmonicolor. The results are presented in table lll below.

TABLE III Dehydrogenation of Octadecane Olefin yield 8.5%

Olefins present cis 9-octadecene 9 l k cis B-octadecene 24% cis 7-octadecene l-2% cis fi-octadecene cis S-octadecene trace EXAMPLE Ill TABLE IV Alkane Alkene yield Percent Dodecane 0.0 Tetradecane 0.5 Pentadecane 2.5 Hexadecane 5 .5 Octadecane 8.5 Eicosane 0.5

The terms and expressions used herein are used as terms of description and not of limitation as there is no intention by the use of such terms and expressions of excluding any equivalent. It is recognized that various modifications and departures in the specific embodiments described herein can be made within the scope of the invention claimed.

We claim:

1. A process of dehydrogenating an alkane which comprises: aerobically contacting Nocardia salmonicolor resting cells with a C,,C alkane under incubation conditions for a suitable period of time to form cis alkenes, separating the resting cells from the hydrocarbons and recovering the alkenes, said Nocardia salmonicolor resting cells having been grown on a carbohydrate-mineral salt medium containing iron salts in a ferrous salt concentration, expressed as the sulfate, of between about 0.0075 and 0.015 wt. percent and a ferric salt concentration, expressed as the sulfate, of between about 0.01 and 0.2 wt. percent.

2. A process according to claim 1 wherein the Nocardia salmonicolar is the strain ATCC 21243.

3. A process according to claim 1 wherein the alkane is hexadecane.

4. A process according to claim 1 wherein the alkane is octadecane.

5. A process according to claim 1 wherein the resting cells are suspended in a liquid selected from the group consisting of distilled water and aqueous phosphate solution buffered at a pH of 7.

6. A process according to claim 1 wherein the incubation conditions comprise:

a. for cell growth,

a temperature of between 26 and 33 C.,

a pH of between 6.8 and 7.2 and a growth period of between 2 and 5 days, and

b. for alkane conversion,

a ratio of resting-cell suspension to alkane of between 1:1

and 5:1,

a temperature of between 23 and 33 C.,

a pH of between 6.6 and 7.8 and a conversion period of between 3 and 7 days.

7. A process according to claim 6 wherein the medium is the DMS nutrient medium of table I, the carbohydrate in the medium is glucose, the ferrous salt concentration is about 0.01 wt. percent and the ferric salt concentration is about 0.1 wt. percent.

8. A process of dehydrogenating an alkane which comprises:

a. aerobically growing Norcardia salmanicolor under incubation conditions on a carbohydrate-mineral salt nutrient medium containing iron salts in a ferrous salt concentratron, expressed as the sulfate, of between about 0.0075

and 0.015 wt. percent and a ferric salt concentration, expressed as the sulfate, of between about 0.01 and 0.2 wt.

percent,

b. separating the N. salmonicolor cells from said medium,

c. preparing a resting-cell suspension of said N. .ralmom'color cells,

d. aerobically contacting said N. salmonicolor resting-cell suspension with a C, C alkane under incubation conditions for a suitable period of time to form cis alkenes,

e. separating the resting cells from the hydrocarbons, and

f. recovering the alkenes.

9. A process according to claim 8 wherein the N. salmonicalor is the strain ATCC 21243.

10. A process according to claim 8 wherein the alkane of step (d) is selected from the group consisting of hexadecane and octadecane.

11. A process according to claim 8 where step (0) comprises suspending the N. salmonicolor cells in a liquid selected from the group consisting of distilled water and an aqueous phosphate solution buffered at a pH of 7.

12. A process according to claim 8 wherein the incubation conditions of step (a) comprise:

a temperature of between 26 and 33 C.,

a pH of between 6.8 and 7.2 and a growth period of between 2 and 5 days and the incubation conditions of step (d) comprise:

a ratio of resting-cell suspension to alkane of between H and 5:1,

a temperature of between 23 and 33 C.,

a pH of between 6.6 and 7.8 and a conversion period of between 3 and 7 days.

13. A process according to claim 12 where in step (a) the medium is the DMS nutrient medium of table 1, the carbohydrate in the medium is glucose, the ferrous salt concentration is about 0.01 wt. percent and the ferric salt concentration is about 0.1 wt. percent. 

2. A process according to claim 1 wherein the Nocardia salmonicolor is the strain ATCC
 21243. 3. A process according to claim 1 wherein the alkane is hexadecane.
 4. A process according to claim 1 wherein the alkane is octadecane.
 5. A process according to claim 1 wherein the resting cells are suspended in a liquid selected from the group consisting of distilled water and aqueous phosphate solution buffered at a pH of
 7. 6. A process according to claim 1 wherein the incubation conditions comprise: a. for cell growth, a temperature of between 26* and 33* C., a pH of between 6.8 and 7.2 and a growth period of between 2 and 5 days, and b. for alkane conversion, a ratio of resting-cell suspension to alkane of between 1:1 and 5:1, a temperature of between 23* and 33* C., a pH of between 6.6 and 7.8 and a conversion period of between 3 and 7 days.
 7. A process according to claim 6 wherein the medium is the DMS nutrient medium of table I, the carbohydrate in the medium is glucose, the ferrous salt concentration is about 0.01 wt. percent and the ferric salt concentration is about 0.1 wt. percent.
 8. A process of dehydrogenating an alkane which comprises: a. aerobically growing Norcardia salmonicolor under incubation conditions on a carbohydratE-mineral salt nutrient medium containing iron salts in a ferrous salt concentration, expressed as the sulfate, of between about 0.0075 and 0.015 wt. percent and a ferric salt concentration, expressed as the sulfate, of between about 0.01 and 0.2 wt. percent, b. separating the N. salmonicolor cells from said medium, c. preparing a resting-cell suspension of said N. salmonicolor cells, d. aerobically contacting said N. salmonicolor resting-cell suspension with a C14-C20 alkane under incubation conditions for a suitable period of time to form cis alkenes, e. separating the resting cells from the hydrocarbons, and f. recovering the alkenes.
 9. A process according to claim 8 wherein the N. salmonicolor is the strain ATCC
 21243. 10. A process according to claim 8 wherein the alkane of step (d) is selected from the group consisting of hexadecane and octadecane.
 11. A process according to claim 8 where step (c) comprises suspending the N. salmonicolor cells in a liquid selected from the group consisting of distilled water and an aqueous phosphate solution buffered at a pH of
 7. 12. A process according to claim 8 wherein the incubation conditions of step (a) comprise: a temperature of between 26* and 33* C., a pH of between 6.8 and 7.2 and a growth period of between 2 and 5 days and the incubation conditions of step (d) comprise: a ratio of resting-cell suspension to alkane of between 1:1 and 5:1, a temperature of between 23* and 33* C., a pH of between 6.6 and 7.8 and a conversion period of between 3 and 7 days.
 13. A process according to claim 12 where in step (a) the medium is the DMS nutrient medium of table I, the carbohydrate in the medium is glucose, the ferrous salt concentration is about 0.01 wt. percent and the ferric salt concentration is about 0.1 wt. percent. 